JPS59204129A - Adjuvant - Google Patents

Abstract

PURPOSE:An adjuvant having high safety, acting for prevention of infectious diseases, consisting of glycoprotein and nitrogen-containing low-molecular-weight substances comprising xylose in a culture of a mycelium belonging to Basidiomyces such as Cortinellus shiitake, etc. cultivated in a solid medium rich in a xylose component as a main component. CONSTITUTION:An adjuvant containing glycoprotein and nitrogen-containing low- molecular-weight substances comprising xylose as a main contained in a culture of a mycelium belonging to Basidiomyces (e.g., Cortinellus shiitake, Pleurotus ostreatus, Flammulina velutipes, pholiota nameko, Lyophyllum aggregatum, etc.) cultivated in a solid medium rich in a xylose component such as bagasse, rice bran, etc. The adjuvant has immune increasing effect on cancers, various kinds of virus diseases, or diseases of immune incompetence (allergic diseases) etc.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to the use of xylose-based glycoproteins and nitrogen-containing low-molecular substances contained in mycelium cultures such as shiitake mushrooms cultured in a solid medium rich in xylose components such as bagasse. The present invention relates to an immune enhancer consisting of:

The relationship between bacterial, viral, and fungal infections and carcinogenesis and the body's immunity is academically clear.

The decline in immune capacity is linked to many infectious diseases, which induces carcinogenesis, and in cancer-bearing states, the decline in immune capacity progresses further.The defense mechanism of the body is complex, and many cells and Substances are involved in a chain.

By the way, the immune system consists of humoral immunity and cell-mediated immunity.
The former mainly involves antigen precipitation by immunoglobulins and clearance by phagocytes, and protects the body from systemic infections, while the latter mainly involves the activation of macrophages by various lymphocytes produced from T-cells. It is involved in the elimination of cell-parasitic antigens.

However, due to aging, drug administration, overwork, etc., the immune system described above declines, weakening the resistance to many infectious diseases and inducing carcinogenesis.Humoral immunity, cell-mediated immunity, or both It is academically clear that it is possible to prevent and treat infectious diseases by preventing a decline in Vaccines that provide attenuated antibodies have generally been developed, but when there is an infection caused by an antigen for which there is no vaccine or a microorganism that is normally considered to be of low virulence, and it is desirable to strengthen overall immunity. Conventionally, there were many substances that had strong side effects or induced anaphylaxis, and had both advantages and disadvantages, but this substance has no side effects and is effective at stimulating humoral immunity and cellular immunity. It is characterized by enhancing both immune capacities. Therefore, regarding viruses such as Hepatitis virus,
Influenza virus, Herpes
Viruses and their effective spectrum are wide.

The immune enhancer according to the present invention is used for cancer and various viral diseases.

In addition, an immune-enhancing effect was observed for immunodeficiency diseases (allergic diseases), etc. In addition, although it is academically unresolved whether the immune enhancer of the present invention enhances immune function through its mechanism of action, it is important to note that the It is inferred that this is because the low-molecular-weight substance increases the number of antibody-producing cells or enhances the ability to produce antibodies. In addition, fungal infections, which are generally said to be of low pathogenicity, are largely caused by a weakened immune system due to debilitating underlying diseases such as malignant tumors and collagen diseases. Some ca
It is also effective against ndida albicans, and the induction of malignant tumors is similar in some respects to fungal infections, so the uses of this substance are extremely wide.

This invention uses as active ingredients sugar, protein, and nitrogen-containing low-molecular substances containing xylose as the main component, which are contained in a mycelium culture cultured in a solid medium rich in xylose components, and are safe. The purpose of the present invention is to provide an immune enhancer that has high anti-inflammatory properties and is effective in protecting against a wide range of infectious diseases.

The present invention will be explained in detail below.

The present inventors first cultivated mycelium belonging to Basidiomycetes, such as shiitake mushrooms, in a solid medium made mainly of fibrous components such as Bacchus and added rice bran, etc., and extracted anticancer substances from this mycelial culture. (Japanese Patent Application Laid-Open No. 57-206618) has completed the invention of an extraction method for extracting the above-mentioned liquid.The present invention was obtained as a result of further research on the effective use of the extract.

That is, as a result of analyzing the above-mentioned extract, it was found that a large amount of glycoproteins and nitrogen-containing low-molecular substances containing xylose as a main component were contained in the extract, and as a result of analyzing the medium components, for example, Bacchus ( Sugar cane dregs), rice bran, etc. contain a large amount of xylose. Therefore, the present inventors believe that sugar, protein, and nitrogen-containing low-molecular substances, mainly composed of xylose, exert their immune-enhancing effects. It was recognized that this was done.

In the present invention, a medium rich in xylose components must be adopted for the above reasons, and examples thereof include monocotyledonous plants such as bagasse, barley straw, rice straw, corn stems and leaves, and cedar; Rice bran is mentioned as a nutritional source.

Furthermore, the basidiomycetes used in the present invention include Shiitake mushrooms, Hirafuki Enoki mushrooms, Nameko mushrooms, Shimeji mushrooms, etc., and the activity extracted from Shiitake mycelium culture was the most excellent.

As a result of analyzing the immune enhancer according to the present invention by gas chromatography, it was found that it contained a glycoprotein mainly composed of xylose and a nitrogen-containing low-molecular substance.

Examples will be described below.

Nutrient sources 5: 90% crushed bagasse, 5% rice bran, bran, etc.
% solid medium is sterilized by a conventional method and inoculated with a solid seed of shiitake mushrooms. The medium after inoculation is kept at room temperature1.
The mycelium is cultured in a culture room air-conditioned at 8°C to 20°C and 60% humidity.

The culture medium on which the above culture has been completed is transferred to a cultivation room and left there.

Shiitake mushroom fruiting bodies then begin to develop from the surface of the medium, and at this point the medium is removed from the cultivation chamber and crushed into toga finger-sized pieces using a crusher.

This pulverized medium (a mixture of mycelium and medium, hereinafter referred to as mycelium culture) is filled into a tank, and the mycelium culture 6009 is
Add the clean water in Step 5 whose pH has been adjusted to 3 to 8, and mix and stir by changing the pH step by step while keeping the pH constant.

In other words, the temperature is varied from 40°C to 60°C according to each pH level, and this is to match the activity conditions of enzymes such as cellulase, chitinase, glucosidase, and protease present in the mycelium culture, and to promote enzymation. be. Finally, the temperature is raised to 60°C to inactivate the enzyme, and mixing and stirring are performed, but this stirring removes mycelial components, mycelial metabolites a'3, and wood decomposition products in the medium components. Leached into water.

The suspension thus obtained is filled into a flannel cloth filter bag, which is then pressurized and filtered, and the filtrate is filtered and sterilized through a membrane filter to obtain an extract.

Next, the extract obtained as described above is freeze-dried to form a powder (hereinafter referred to as LEM).

Example (1) Implementer: Department of Biology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University (
Presented at the 103rd Annual Meeting of the Pharmaceutical Society of Japan) <2) 5 epl + aro of the alcohol precipitated part of LEM
Fractionation was performed using se6B to obtain two peaks, which were labeled LAP1 and LAP2 from the void side.

(3) Macrophages Macrophages of peritoneal exudate cells induced from guinea pigs (4) Proteins of rat ascites liver cancer (A84.14) (5)
Experimental method LAPl and LA P2 were intraperitoneally administered to Donryu rats from the day before transplanting Al-1414, and lli! The viscera is removed and transformed into cells. A glass capillary is layered with guinea pig macrophages, spleen cells, and an antigen protein solution, and after culturing in a petri dish for 48 hours, the area created by migrating cells is measured.

(6) In the LAP1 administration group, macrophage migration activity was observed for 4 to 60 days after administration, but no activation was evident in LA1]2. In addition, in the photograph substituted for the reference drawing, the left side shows the LΔP1 administration group, and the right side shows the LAP2 administration group.

LAPl is Δrabinose, G 1ucose,
Qalactose.

50-8 consisting of a polysaccharide composed of M annoselZ ylose and a peptide mainly composed of produced amino acids
It turned out to be vepzidoglucan with a molecular weight of 1,000,000.

Example (1) Implementer Osaka City University School of Medicine, Department of Biochemistry (2) In cellular immunity, antibody-producing cells are in vitro treated with non-specific lymphocyte activators (juvenile lymphocytes). Propagation follows the same process.

When lymphocytes are cultured in the presence of PWM, they become B1asvcells and DNA synthesis is renewed. Therefore, by adding H3-thymicline as a DNA precursor, culturing the cells, and measuring the H3-thymidine taken into the cells, the degree of lymphocyte activation can be determined.

(3) Lymphocytes Human peripheral blood lymphocytes (4) Experimental method PWM and LEM were added to the lymphocyte suspension and cultured for 96 hours. Next, 31-(-thymidtne was added and cultured for 24 hours. The cells were then cultured for 24 hours. It was dissolved, and the incorporation of 3 H-thymidine was measured using a cinderase counter.

(5) Results LEM is DNA synthesis by PWM.
It was promoted in the range of 100 to 300 μg/mIA. In addition, LEM alone has the effect of promoting DNA synthesis.
ymphocyte blastogenic fac
It was confirmed that it was tor.

Example (1) Performer Osaka City University School of Medicine, Department of Biochemistry 2) Experiments related to antibody production (3) Experimental method LEM was added to macrophages immunized with TNP-3RBC and cultured for 24 hours. The supernatant was taken, human peripheral blood cells were added together with PWM, and the production of anti-TNP-3RBC was confirmed by the plague method.

LEM promotes the production of antibodies against TNP-8RBC by P'WM, and its optimal degree is 200 μq/m Jl.
Met. In addition, a small amount of antibody production activity was observed in EM itself.

Example (1) Human peripheral blood C0nCa1laVa l 1ll
1nter biosynthesized by stimulating culture with A and LEM
Quantifying the production amount of Ienkin-1 By adding 50 μg/m or more of LEM to C0DA, the production of IL-1 was significantly increased.

Next, the LEM was fractionated using 5 ephadex G-75 to obtain three fractions (Fl from void, F2°[:3) showing ultraviolet absorption. (See Figure 2.) Next, the above F1. F2. Interlen of each F3 fraction
Kin-1 assay was performed.

F3 N19 child ff110.000g close to Votal
.

Activity similar to that of nitrogen-containing compounds (see below) was observed.

Example (1) Implementer: Meiji Pharmaceutical Humanities and Microbiology (2) The effect of LEM on fungal infections in an immunosuppressed state was confirmed.

(3) Animal fruit M mouse fcRK () 6-7 weeks old (4) Attack tube candida albicans
(J-101210”/molts (3) (5) Immunosuppressant cOrtiSOne (6) LEM administration Administration method and route 300μ (7/m.

use intraperitoneal administration (7) Experimental group M4 adult (8) M fruit Q, survival rate 25 days after albicans inoculation G-125% G-II 83% G-I111
00% G-IV50% or more, EM has been recognized to be effective in preventing infection in mice in an immunosuppressed state, and its administration does not require long-term administration, and can be administered after bacterial infection. It has been found to be fully effective.

[Brief explanation of the drawing]

The first is a developed image showing the state in which the material according to the present invention is developed using Sephadex G75. Procedural amendment 1. Indication of the case Patent application No. 5858-7914 5. Title of the invention: Immune enhancer 3. Person making the amendment Relationship to the case Patent applicant Address: 121 Shimizu, Noda City, Chiba Prefecture Name: Noda Shokubaku Kogyo Co., Ltd. Representative name: Iizuka Chiyokichi 4, Agent 101 Address 1-15-16-6 Uchikanda, Chiyoda-ku, Tokyo
, Full text of the specification to be amended and drawings 2. Specification 1. Name of the invention Immune enhancer 2. Claims (1) Mycelium belonging to Basidiomycetes such as Shiitake mushrooms cultured in a solid medium rich in xylose component An immune enhancer consisting of sugars, proteins, and nitrogen-containing low-molecular substances mainly consisting of xylose in culture. (2) The immune enhancer according to claim 1, wherein the solid medium comprises bagasse and rice bran. (3) The immune enhancer according to claim 1, wherein the basidiomycete is a shiitake mushroom. 3. Detailed Description of the Invention The present invention relates to sugars, proteins, and nitrogen-containing substances mainly containing xylose contained in mycelial cultures of shiitake mushrooms, etc., cultured in a solid medium rich in bagasse and xylose components. This invention relates to an immune enhancer consisting of a molecular substance. The relationship between infectious diseases and carcinogenesis caused by Ill bacteria, viruses, and fungi and the immune system of the body is academically clear. The decline in immune function is linked to many infectious diseases, induces cancer development, and in cancer-bearing states, the decline in immune function progresses further.The body's defense mechanism is complex, and many cells and substances exist in body fluids. are involved in a chain reaction. By the way, the immune system consists of humoral immunity and cell-mediated immunity.
The former mainly involves antigen precipitation by immunoglobulins and clearance by phagocytes, and protects the body from systemic infections, while the latter involves the activation of macrophages by various lymphocytes produced from 1-cell. The main effect is
Involved in eliminating cell-parasitic antigens. However, due to aging, drug administration, overwork, etc., the immune system described above declines, weakening the resistance to many infectious diseases and inducing carcinogenesis.Humoral immunity, cell-mediated immunity, or both It is academically clear that preventing and treating infectious diseases is possible by preventing a decline in Vaccines that provide attenuated antibodies have generally been developed, but when there is an infection caused by an antigen for which there is no vaccine or a microorganism that is normally considered to be of low virulence, and it is desirable to strengthen overall immunity. Conventionally, there were many substances that had strong side effects or induced anaphylaxis, and had both advantages and disadvantages, but this substance has no side effects and is effective against humoral immunity J3 and cellular immunity. It is characterized by enhancing the immune capacity of both. Therefore, regarding viruses, Hepatitis virus 11
nfluenza virus, Hcrpesv
irus and its effective spectrum is wide. The immune enhancer according to the present invention is used for cancer and various viral diseases. In addition, an immune-enhancing effect was observed for immunodeficiency diseases (allergic diseases), etc. In addition, although it is academically unresolved by what mechanism of action the immunopotentiator of the present invention enhances immune function, it is important to note that the It is inferred that this is because the low-molecular-weight substance increases the number of antibody-producing cells or enhances the ability to produce antibodies. 1. Fungal infections, which are generally said to be of low pathogenicity, are largely caused by a weakened immune system due to debilitating underlying diseases such as malignant tumors and collagen diseases; It is also effective against the fungus Candida albicans,
Since the induction of malignant tumors is similar in some respects to fungal infections, the uses of this substance are extremely wide. This invention uses as active ingredients sugar, protein, and nitrogen-containing low-molecular substances containing xylose as the main component, which are contained in a mycelium culture cultured in a solid medium rich in xylose components, and are safe. The purpose of the present invention is to provide an immune enhancer that has high anti-inflammatory properties and is effective in protecting against a wide range of infectious diseases. The present invention will be explained in detail below. The present inventors first cultivated mycelium belonging to Basidiomycetes, such as shiitake mushrooms, in a solid medium made mainly of fibrous components such as Bakasu and to which rice bran, etc. were added. Although he has completed the invention of an extraction method for extracting substances (
(Japanese Patent Application Laid-Open No. 57-206618), the present invention was obtained as a result of developing the above-mentioned invention and further researching the effective use of the extract. That is, as a result of analyzing the above-mentioned extract, it was found that the extract contained large amounts of sugars, proteins, and nitrogen-containing low-molecular substances whose main component was xylose, and as a result of analyzing the medium components, for example, Bacchus ( Xylose is contained in large amounts in sugar cane dregs), rice bran, etc. Therefore, the present inventors believe that sugar, protein, and nitrogen-containing low-molecular substances, mainly composed of xylose, exert their immune-enhancing effects. It was recognized that this was done. In the present invention, for the above reasons, a medium rich in xylose components must be used, and examples thereof include monocots such as bagasse, wheat straw, rice straw, corn stems and leaves, and cedar; Rice bran is mentioned as a nutrient source for the culture medium. Furthermore, examples of the basidiomycete used in the present invention include shiitake mushrooms, hirabuki mushrooms, enoki mushrooms, nameko mushrooms, shimeji mushrooms, etc., and the activity extracted from shiitake mycelium culture was the most excellent. As a result of analyzing the immunopotentiator according to the present invention by gas chromatography, it was found that it contained sugars whose main components were xylose, proteins, and nitrogen-containing low-molecular substances. Examples will be described below. Nutrient sources 5: 90% crushed bagasse, 5% rice bran, bran, etc.
% solid medium is sterilized by a conventional method and inoculated with a solid seed of shiitake mushrooms. The medium after inoculation is kept at room temperature1.
The mycelium is cultured in a culture chamber air-conditioned at 8°C to 2C1 and 60% humidity. The medium on which the above culture has been completed is transferred to a cultivation room and left there. Shiitake mushroom fruiting bodies then begin to develop from the surface of the medium, and at this point the medium is removed from the cultivation chamber and crushed into toga finger-sized pieces using a crusher. This pulverized medium (a mixture of mycelium and medium, hereinafter referred to as mycelial culture) is filled in a tank, and at the same time, the pH of 600 g of mycelial culture is 3 to 8. Add fresh water adjusted to 5°C, and mix and stir by changing the temperature in stages at a constant time. In other words, the temperature is varied from 40°C to 60°C according to each pH level, and this is to match the activity conditions of enzymes such as cellulase, gitinase, glucosidase, and protease present in the mycelium culture and to promote enzymation. be. Finally, the temperature was raised to 60°C to also deactivate the enzyme.
Mixing and stirring are carried out by raising the temperature to a certain temperature, and by this stirring, mycelium components, mycelium metabolites, and wood decomposition products in the medium components are leached into water. The suspension thus obtained is filled into a flannel cloth filter bag, which is then pressurized and filtered, and the filtrate is filtered and sterilized through a membrane filter to obtain an extract. Next, the extract obtained as described above is freeze-dried to form a powder (hereinafter referred to as LEM). Example (1) Implementer: Department of Biology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University (
(Presentation at the 103rd Annual Meeting of the Pharmaceutical Society of Japan) (2) The alcohol precipitated portion of LEM was
Fractionation was performed using e6B to obtain two peaks, which were labeled LAP1 and LAP2 from the void side. (3) Macrophages Macrophages of peritoneal exudate cells induced from guinea pigs (4) Proteins of rat ascites liver cancer (AH414) (5) Experimental method LAP1 and LA P2 were intraperitoneally administered from the day before transplanting Al-1414 to Donryu rats. and Al-1414 transplantation 1 2・-8
The spleen is removed and transformed into cells every day. Guinea pig macrophages, spleen cells, and an antigen protein solution are layered on a glass capillary tube, and after culturing in a petri dish for 48 hours, the area created by the migrated cells is measured. (6) In the LAP1 administration group, macrophage migration activity was observed between 4 and 6 days after administration, but no activation was evident in LAP2. In addition, in the photograph substituted for the reference drawing, the left side shows the LAP1 administration group, and the right side shows the LAP2 administration group. L A P 1 is A rabinose, Q 1uc
ose, Qalactose. 50-80 consisting of polysaccharides composed of Mannose and xylose and peptides mainly composed of produced amino acids
It turned out to be a peptidoglucan with a molecular weight of 1,000,000. Example (1) Implementer Osaka City University School of Medicine, Department of Biochemistry (2) In cellular immunity, antibody-producing cells undergo the same process as lymphocyte juvenileization in vitro by non-specific lymphocyte activators. Follow and propagate. When lymphocytes are cultured in the presence of PWM, they become BlasVcells and DNA synthesis is renewed. Therefore, the degree of activation of lymphocytes can be determined by adding H3-thymidine as a DNA precursor, culturing the cells, and measuring H3-thyn+1dine taken into the cells. (3) Lymphocytes Human peripheral blood lymphocytes (4) Experimental method PWM iJ3 and LEM were added to the lymphocyte suspension and cultured for 96 hours, then 3H-thymidine was added and cultured for 24 hours. Cells were lysed, and 3 H-thymidine uptake was measured using a scintillation counter. (5) Results LEM is DNA synthesis by PWM.
It was promoted in the range of 11 degrees 100 to 300μ9/m. Furthermore, EM alone has the effect of promoting DNA synthesis, and LEM has the effect of promoting DNA synthesis.
mphocyte blastogenic fact
It was confirmed that or. Example (1) Implementer Osaka City Faculty of Human Sciences, Department of Biochemistry 2 (2) Experiment on antibody production (3) Experimental method LEM was added to macrophages immunized with TNP-8RBC and cultured for 24 hours. The culture supernatant was taken, human peripheral blood cells were added together with PWM, and production of anti-TNP-8RBC was confirmed by the plaguC method. Is LEM compatible with TNP-8RBC using PWM? The optimal concentration was 200 μQ/If. Furthermore, antibody production activity was observed in LEM itself, even though it was f'X. Example (1) E: Concanavali peripheral blood
nA and LEM tea stimulation culture L/shi biosynthesis sample 8int
Quantitatively quantify the production amount of er 1eukin-1 to C0nA: Ll:M3C1[]/m 1. By adding the above, the production of IL-1 increases significantly and IC. Next, the LEM was fractionated with 5ephadexG-7'5 and showed ultraviolet absorption. I'3'') fraction (void
From this, Fl, F2° (:3) was obtained. (See Figure 2.) Next, the above F1. F2. InterLeL of each F3 fraction
An Ikill-(assay was performed. An activity close to that of F3 (a nitrogen-containing compound with an estimated molecular weight of 10,000 or less) close to Total was observed. Example (1) Implementer Meiji Pharmaceutical University Microbiology Department (2) Immunosuppression (3) Animal experiment Mouse rcRK () 6-7 weeks old (4) Attacking bacteria Candida albicans
(J-1012105/mouse (5) Immunosuppressant Cortisone (6) LEM administration and route 300 μg/m. Use intraperitoneal administration (7) Experimental group composition (8) Results Survival rate 25 days after C. albicans inoculation G-125% G-[I83% G-I[[100%
G-IV of 50% or higher indicates that LEM is effective in preventing infection in immunosuppressed mice, and its administration does not require long-term administration, and is sufficiently effective even when administered after bacterial infection. It turns out that it does. Example (1) Purpose LEM polyclonal act
Examine ivator activity. (2) Experimenter: Department of Biochemistry, Osaka City School of Human Sciences (3) Experimental method: Mononuclear cells rich in lymphocytes were separated from normal human peripheral blood by l"1col l-Conray multilayer centrifugation, and 10% immobile. A cell suspension of 2X 106 cells/m was made with Eagle MEM containing fetal bovine serum. Various concentrations of LEM were added to this and cultured at 37°C for 72 hours to determine the production of anti-TPN-8RBC antibodies. The number of cells was measured by plaque counting. (4) Results As shown in Fig. 2, 100 to 2
By adding 00μ+1/vU of LEM, the number of antibody-producing cells was significantly increased compared to the control group to which LEM was not added. Although this is weak against LEM, polyclo, na
activator activity. Example (1) Purpose To examine the effect of LEM on antibody production of PWM-stimulated peripheral blood mononuclear cells. (2) Experimenter: Department of Biochemistry, Faculty of Medicine, Osaka Jioki University (3) Experimental method: Mononuclear cell suspension prepared from normal human peripheral blood (
Pokewe to 2X 10' cells /,g,)
ed mitogen (PWM) at 50μgz”
n+u and at the same time various concentrations of LEM were added and cultured at 37°C for 72 hours, and the number of anti-TNP-8RBC antibody producing cells was measured by plaque counting. (4) Results Resistant NP-8 in the system without adding LEM
The number of RBC antibody-producing cells is 10'viable
The total number of cells per cell was 632±109. L[EM
The number of anti-TNP-8RBC antibody producing cells in the system in which LEM was added is expressed as a percentage of the system in which LEM was not added.
It became as shown in g3. That is, antibody production was significantly promoted, especially when 200 μg/m of EM was added.
The number of anti-TNP-8RBG antibody-producing cells was about 1.7 times that of the system without LEM. From this result, it is considered that LEM acts on either lymphocytes or monocytes involved in antibody production to amplify antibody production. Example (1) Purpose To examine the effect of ELM-treated macrophage culture supernatant on lymphocyte proliferation. (2) Experimenter: Department of Biochemistry, Faculty of Medicine, Osaka Jooki University (3) Experimental method: Macrophages of guinea pig peritoneal exudate cells were cultured with various concentrations of LEM for 6 hours, and the culture supernatant was separated. On the other hand, normal human peripheral blood mononuclear cell suspension (IX106cel
ls/mu), and added 0.1 ml of the above-mentioned l-2M-treated macrophage culture supernatant to the 17IIl, and after culturing for 48 hours, the specific activity of 3H-thymidine was increased to 5Ci/mu.
mmol) was added at 1 uci/m and incubated for an additional 24 hours. Then, lymphocyte proliferation was measured using the incorporation of 31''-thymidine into the acid-insoluble fraction as an indicator. In addition, macrophage culture supernatants treated with various concentrations of LEM in the same manner as described above were added to a normal human peripheral blood mononuclear cell suspension together with PWM, and after 48 hours of culture, 3H
-Thymidine was added and cultured for 24 hours, and lymphocyte proliferation was measured. (4) Results In the system without PWM, Fio4
As shown in , Lf of 100-200μm/11III
In the macrophage culture supernatant treated with EM, an activity that significantly promoted the proliferation (DNA synthesis) of mononuclear cells was observed. From this, it is inferred that activation of macrophages may be partially involved in polyclonal antibody production of LEM shown in f-ig2. In addition, regarding the proliferation of PWM-stimulated mononuclear cells, F1(
15 days CT-U: 50-3ooμg/lEM
When LEM-treated macrophage culture supernatant was added, lymphocyte proliferation was approximately 1.5 times higher than when LEM-untreated macrophage culture supernatant was added. From this, PW
It is speculated that antibody production by M-stimulated mononuclear cells and amplification by EM is due to macrophage activation. However, a small amount of L is present in the macrophage culture supernatant.
Since it contains EM, the possibility that LEM acts directly on lymphocytes cannot be completely denied. However, since only 0.1 frog was added to the macrophage culture supernatant per mononuclear cell suspension, it is thought that even if LEM acted directly on lymphocytes, the extent of the effect would be low. Example (1) Purpose To investigate the fractionation of 12M-treated macrophage culture supernatant and the effect of each fraction on lymphocyte proliferation. (2) Experimenter: Department of Biochemistry, Faculty of Medicine, Osaka Jooki University (3) Experimental method: The culture supernatant of macrophages treated with LEM was fractionated using a 5 ephadex Q column (5 x 15'Ocm), and the protein was eluted from the absorbance at 280 nm. When the curve is created, it will look like the one shown in Fig.6. From this elution pattern, divide the eluate into 11g7 portions,
After correcting the concentration to the volume before applying it to the column, 0.1 ml of each was added to 1 ml of mononuclear cell suspension (2x, 106 cells/ml) along with ConA (20μo/m), Experimental Example 8 In the same manner as above, lymphocyte proliferation was measured using the incorporation of 3H-zymidine into the acid-insoluble fraction as an index. (4) Results As shown in 11g7, in the l-E M-treated macrophage culture supernatant addition group, DNA synthesis of lymphocytes by ConA was markedly increased, and in particular, when the fifth fraction was added, the DNA synthesis of lymphocytes was increased by about 4 times. became. This suggests the possibility that interleukin 1 exists in the LEM-treated macrophage culture medium. Example (1) Purpose To examine the effect of LEM on isolated hepatocyte injury caused by activated macrophages. (2) Experimenter: Department of Biochemistry, Faculty of Medicine, Osaka Juiki University (3) Experimental method: Isolated hepatocyte suspension (1x10cells/
vn) was treated with various concentrations of LEM for 6 hours. 3H-
The amount of protein synthesis in hepatocytes was measured using the incorporation of leucine acid into the acid-insoluble fraction as an index. (4) Results When hepatocytes were previously treated with 200 μg/Makoto's LEM and then activated macrophage culture supernatant was added, hepatocyte protein synthesis decreased. This result suggests that LEM has a protective effect on hepatocytes. 4. Brief explanation of the drawings Fig. 1 is a fractionation development diagram showing the state in which the substance according to the present invention is developed using Sephadex G75, and Fig. 2 is a diagram showing the activity of polyclonal activator of LEM using a graph. , Figure 3 is a graph showing the effect of LEM on antibody production of PWM-stimulated peripheral blood mononuclear cells, and Figure 4 shows that L'EM-treated i-macrophage culture supernatant had no effect on lymphocyte proliferation. Figure 5 shows the P of 12M-treated macrophage culture supernatant.
A diagram showing the effect on WM-stimulated mononuclear cell proliferation using a graph, Figure 6 is a fractionation development diagram of LEM-treated macrophage supernatant using Sephadex G-75 column chromatography,
Figure 7 shows Con of both LEM-treated macrophage supernatants;
FIG. 8 is a graph showing the effect of LEM on 61-stimulated mononuclear cell proliferation, and FIG. 8 is a graph showing the protective effect of LEM against hepatocyte damage caused by the culture supernatant of activated macrophages. Patent Applicant Noda Shokuboku Kogyo Co., Ltd. Figure 2 C0NCENTRArlQN Figure 3 C0NCENTRA'rLON Figure 4 C0NCENTRA'rLON Figure 5 CONCENrRATjON Figure 6 TUBE NUMBER Figure 7

Claims (1)

[Scope of Claims] (1) Consisting of glycoproteins containing xylose as a main component and nitrogen-containing low-molecular substances in a mycelium culture belonging to Basidiomycetes such as shiitake mushrooms cultured in a solid medium rich in xylose components. Immune enhancer. (2) The immune enhancer according to claim 1, wherein the solid medium comprises bagasse and rice bran. (3) The immune enhancer according to claim 1, wherein the basidiomycete is a shiitake mushroom. The immune enhancer according to item 1.